Oligonucleotide Chip for Detection of Lamivudine-Resistant Hepatitis B Virus

Abstract

Hepatitis B virus (HBV) is one of the major causes of liver disease worldwide. It is important to conduct antiviral therapy against chronic hepatitis B to minimize the amount of liver damage. Lamivudine has been known to be an effective antiviral agent for the treatment of HBV infection. However, the emergence of viral mutants resistant to lamivudine is the main concern during the treatment of HBV-infected patients. Therefore, the detection of lamivudine-resistant mutants is of clinical importance. We have developed an oligonucleotide chip for the detection of lamivudine-resistant HBV which is rapid and accurate. The oligonucleotide chip consists of quality control probes, negative control probes, and specific oligonucleotide probes for the detection of lamivudine-resistant HBV. The specific probes consist of five probes for the detection of wild-type rtL180, rtM204, and rtV207 sequences and seven probes for the detection of HBV mutations. We tested 123 serum samples from patients with chronic HBV infection who had received lamivudine therapy. Eighty samples contained mutants with YMDD mutations. Among these, 17 contained rtM204V (YVDD), 24 contained rtM204I3 (YIDD3), 3 contained rtM204I2 (YIDD2), and 36 contained mixed types. We compared the results obtained with our oligonucleotide chip with those obtained by PCR-restriction fragment length polymorphism (RFLP) analysis and sequencing. The rate of concordance between the assay with the oligonucleotide chip and PCR-RFLP analysis for detection of the YMDD motif was 96.7%. The rate of concordance between the results obtained with the oligonucleotide chip for the detection of rtL180 and rtV207 and the results obtained by sequencing was 100%. Thus, the oligonucleotide chip is a reliable and useful tool for the detection of antiviralresistant HBV.Hepatitis B virus (HBV) is one of the major causes of liver disease worldwide, and chronic hepatitis B (CHB) can progress to cirrhosis and hepatocellular carcinoma. It is important to conduct antiviral therapy against CHB to minimize the amount of liver damage (15). The development of nucleotide analogs which inhibit HBV reverse transcriptase activity, such as lamivudine, famciclovir, and others, has provided an alternative to interferon for therapy for CHB. Lamivudine, (Ϫ)-␤-L-2Ј,3Ј-dideoxy-3Ј-thiacytidine, is a known inhibitor of RNA-dependent DNA polymerase of HBV and human immunodeficiency virus (2, 12, 18). Lamivudine treatment of patients with CHB has been shown to be effective in suppressing virus replication and to result in reduced inflammatory activity (1, 2, 8, 9, 17). However, prolonged lamivudine therapy has been associated with increased rates of emergence of lamivudine-resistant HBV. The cause of lamivudine-resistant HBV was revealed to be the amino acid substitutions from leucine to methionine at codon 180 of the B domain (rtL180M) and amino acid substitutions of the YMDD motif from methionine to valine or leucine at codon 204 of the C domain (rtM204V or rtM204I) of the reverse transcriptase (rt) region of th...

Evaluation of antiviral resistant hepatitis B virus subpopulations in patients with chronic hepatitis B by using terminal restriction fragment length polymorphism

“…In this context, for the genotypic evaluation of antiviral resistant hepatitis B virus subpopulations, various monitoring methods have been developed as alternatives to commonly used direct DNA sequencing method. These include, PCR-RFLP method [1,7], PCR-RFMP (restriction fragment mass polymorphism) method [4], methods using conventional PCR with allele specific primers [10,19,20], peptide nucleic acid (PNA) mediated PCR clamping method [8], methods based on real-time PCR [3,5,12,13,17,18] and methods based on immobilized oligonucleotide probes [6,9,11,15,21,22]. The sensitivity of these methods in detecting antiviral resistant HBV subpopulations has been reported to be 5 % in PCR-RFLP method [1], 1 % in PCR-RFMP method [4], 0.5 % in allele specific PCR method [10], 0.01 % in PNA mediated PCR clamping method [8], depending on the experimental settings 10-0.01 % in real-time PCR methods [3,5,12,13,17,18] and 30-5 % in methods using immobilized oligonucleotide probes [6,9,11,21,22].…”

Peptide Nucleic Acid Array for Detection of Point Mutations in Hepatitis B Virus Associated with Antiviral Resistance

“…In recent years, various methods for the detection of antiviral resistance have been developed and applied (8,16,21). Microarray-based techniques have become well established as powerful tools for the diagnosis and monitoring of infectious diseases (5,13,22,28).…”

“…Direct sequencing can identify known and potential new resistance mutations, but it is a time-consuming and laborious method. Furthermore, sequencing can detect minor populations comprising more than 20% of the total virus population (8,13,26). In particular, the sequencing method is able to correctly determine the nucleotide mixture at position 1 (R ϭ A or G) and position 3 (K ϭ T or G, R ϭ A or G, and S ϭ C or G) of codon 204 (11,23).…”

“…Other methods have been used to overcome some of the limitations of sequencing, including PCR restriction fragment length polymorphism (RFLP) analysis and reverse hybridization (line probe assay and oligonucleotide array). However, these methods have disadvantages in terms of specificity, sensitivity, operational complexity, and interpretation of results (1,8,13,17). Most hybridization methods usually make use of a DNA probe, which can be a synthetic oligonucleotide or longer enzymatically generated DNA.…”

“…The sensitive and early detection of emerging resistance is important for monitoring the viral dynamics associated with treatment and to improve therapeutic decision making. Consequently, the method for the detection of antiviral-resistant HBV must be rapid, sensitive, and accurate for reliable diagnosis (13,21).…”